PROJECT SUMMARY/ABSTRACT Our goal is to define how lymphatic molecules control AQH drainage and intraocular pressure (IOP) elevation. IOP elevation is a major risk factor for glaucoma, a disease that will affect 80 million people by the end of the decade. Glaucoma therapy is based on reducing elevated IOP but no current drug is able to reduce IOP very effectively indicating the pressing need for improved therapies. IOP elevation results from resistance to aqueous humor (AQH) drainage, which occurs in the vicinity of the inner wall endothelium of Schlemm's canal (SC). However, the molecular mechanisms functioning in the SC inner wall cells, and in generating resistance to AQH drainage, are not well defined. The SC of mice and humans are very similar anatomically and with respect to AQH outflow physiology; thus the mouse is a powerful model for studying SC function. Using the mouse, we showed that SC is a unique vessel with both blood endothelial and lymphatic endothelial features. A key feature of SC is expression of the lymphatic master regulator transcription factor Prox1, which is required for lymphatic development and maintenance. Based on our previous study showing that PROX1 is enriched in SC inner wall cells and is likely important for functional specialization of these cells, and on known functions of PROX1 in lymphatic tissues, Prox1 is a strong candidate for our functional studies. In this project we will determine the role of Prox1 in controlling AQH drainage. We will accomplish this in three aims. Aim 1) To determine if Prox1 haploinsufficiency disrupts AQH outflow and raises IOP. In preliminary studies using mice we show that Prox1 heterozgosity in the SC elevates IOP. We will use this tool in Aim 1 to define for the first time the role of Prox1 in regulating AQH drainage and IOP. Aim 2) Determine the effect of Prox1 knockout on SC development and function. To accomplish this, Prox1 will be deleted conditionally in mice at critical stages of SC development. These experiments will allow identification of potentially new morphogenetic functions for Prox1 in the developing SC and comprehensive determination of PROX1 function in the adult SC. Aim 3) Identify Prox1-regulated pathways in SC that control AQH outflow or IOP. Prox1-regulated genes in the SC inner wall are candidates for controlling AQH outflow. To identify candidate genes we will use RNAseq and differential expression analysis as well as targeted proteomics to define Prox1 haploinsufficiency-induced changes in SC that cause IOP elevation. Using these data, candidate pathways and hub genes important for AQH outflow will be identified. Importantly, our project will use innovative new approaches that we developed for high-resolution examination of the mouse SC using the Prox1-GFP mouse and other fluorescent genetic reporters and accurate measurement of AQH outflow in the mouse. Successful completion of these aims will yield critical new information on the mechanisms regulating IOP, and will lay the groundwork for identification of more effective therapeutic targets for glaucoma.